Cell Navigator® Mitochondrion Staining Kit *Orange Fluorescence*

Protocol summary

1. Prepare cells
2. Add Mitolite™ Orange working solution
3. Incubate at 37°C for 30 minutes to 2 hours
4. Analyze the cells under fluorescence microscope at Ex/Em = 540/590 nm (TRITC filter set)

Important notes
Thaw all the components at room temperature before starting the experiment.

20 µL of 500X Mitolite™ Orange (Component A) into 10 mL of Live Cell Staining Buffer (Component B) to make Mitolite™ Orange working soluttion. Protect from light. Note:  20 µL of 500X Mitolite™ Orange (Component A) is enough for one 96-well plate. The optimal concentration of the fluorescent mitochondrial indicator varies depending on the specific application. The staining conditions may be modified according to the particular cell type and the permeability of the cells or tissues to the probe.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

For adherent cells:

1. Grow cells either in a 96-well black wall/clear bottom plate (100 µL/well/96-well plate) or on cover-slips inside a petri dish filled with the appropriate culture medium.
   
   
2. When cells reach the desired confluence, add equal volume of Mitolite™ Orange working solution.
   
   
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 minutes to 2 hours.
   
   
4. Replace Mitolite™ Orange working solution with Hanks and 20 mM Hepes buffer (HH buffer) or buffer of your choice (e.g. the buffer with growth medium at 1:1 concentration).
   
   
5. Observe the cells using a fluorescence microscope with TRITC filter set (Ex/Em = 540/590 nm). Note: It’s recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained.

For suspension cells:

1. Centrifuge the cells at 1000 rpm for 5 minutes to obtain a cell pellet and aspirate the supernatant.
   
   
2. Resuspend the cell pellets gently in pre-warmed (37°C) growth medium, and add equal volume of Mitolite™ Orange working solution.
   
   
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 minutes to 2 hours.
   
   
4. Replace Mitolite™ Orange working solution with Hanks and 20 mM Hepes buffer (HH buffer) or buffer of your choice (e.g. the buffer with growth medium at 1:1 concentration).
   
   
5. Observe the cells using a fluorescence microscope with TRITC filter set (Ex/Em = 540/590 nm). Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained. Suspension cells may be attached to cover-slips that have been treated with BD Cell-Tak^® (BD Biosciences) and stained as adherent cells.